Although de novo synthesis of cholesterol occurs in virtually all cells, this capacity is greatest in liver, intestine, adrenal cortex and reproductive tissues, including ovaries, testes and placenta. From an inspection of its structure, it is apparent that cholesterol biosynthesis requires a source of carbon atoms and considerable reducing power to generate the numerous carbon-hydrogen and carbon-carbon bonds. All of the carbon atoms of cholesterol are derived from acetate. Reducing power in the form of NADPH is provided mainly by enzymes of the hexose monophosphate shunt, specifically, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The mevalonate pathway, i.e., the pathway of cholesterol synthesis, occurs in the cytoplasm and is driven in large part by the hydrolysis of the high-energy thioester bonds of acetyl CoA and the high-energy phosphoanhydride bonds of ATP. For a detailed discussion of the mevalonate pathway, see, e.g., Stryer, L., BIOCHEMISTRY, Third Edition (W.H. Freeman And Company/New York (1988)).
The first committed step in the mevalonate pathway is the synthesis of mevalonic acid, which is derived from acetyl CoA. Acetyl CoA can be obtained from several sources: (a) the .beta. oxidation of long-chain fatty acids; (b) the oxidation of ketogenic amino acids such as leucine and isoleucine; and (c) the pyruvate dehydrogenase reaction. In addition, free acetate can be activated to its thioester derivative at the expense of ATP by the enzyme acetokinase, which is also referred to as acetate thiokinase.
The first two steps in the mevalonate pathway are shared by the pathway that also produces ketone bodies. Two molecules of acetyl CoA condense to form acetoacetyl CoA in a reaction catalyzed by acetoacetyl CoA thiolase (acetyl CoA: acetyl CoA acetyltransferase). The next step introduces a third molecule of acetyl CoA in the cholesterol pathway and forms the branched-chain compound 3-hydroxy-3-methylglutaryl CoA (HMG CoA). This condensation reaction is catalyzed by HMG CoA synthase (3hydroxy-3-methylglutaryl CoA: acetoacetyl CoA lyase). Liver parenchymal cells contain two isoenzyme forms of HMG CoA synthase; one is found in the cytosol and is involved in cholesterol synthesis, while the other has a mitochondrial location and functions in the pathway that forms ketone bodies. In the HMG CoA synthase reaction, an aldol condensation occurs between the methyl carbon of acetyl CoA and the .beta.-carbonyl group of acetoacetyl CoA with the simultaneous hydrolysis of the thioester bond of acetyl CoA.
The step that produces the unique compound mevalonic acid from HMG CoA is catalyzed by the important microsomal enzyme HMG CoA reductase (mevalonate: NADP+oxidoreductase) that has an absolute requirement for NADPH as the reductant. This reduction reaction is irreversible and produces (R)-(+) mevalonate, which contains six carbon atoms. HMG CoA reductase catalyzes the rate-limiting reaction in the pathway of cholesterol biosynthesis. HMG CoA reductase is an intrinsic membrane protein of the endoplasmic reticulum whose carboxyl terminus extends into the cytoplasm and carries the enzyme's active site.
Mevalonate is converted into 3-isopentenyl pyrophosphate by three consecutive reactions involving ATP. In the last step, the release of CO.sub.2 from 5-pyrophosphomevalonate occurs in concert with the hydrolysis of ATP to ADP and P.sub.i. Thereafter, squalene is synthesized from 3-isopentenyl pyrophosphate by the reaction sequence: EQU C.sub.5 .fwdarw.C.sub.10 .fwdarw.C.sub.15 .fwdarw.C.sub.30
This stage in the mevalonate pathway starts with the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate. These isomeric C.sub.5 units condense to form a C.sub.10 compound: an allelic carbonium formed from dimethylallyl pyrophosphate is attacked by isopentenyl pyrophosphate to form geranyl pyrophosphate. The same kind of reaction occurs again: geranyl pyrophosphate is converted into an allelic carbonium ion, which is attacked by isopentenyl pyrophosphate. The resulting C.sub.15 compound is called farnesyl pyrophosphate. The last step in the synthesis of squalene is a reductive condensation of two molecules of farnesyl pyrophospate.
The final stage of the mevalonate pathway starts with the cyclization of squalene. This stage, in contrast to the preceding ones, requires molecular oxygen. Squalene epoxide, the reactive intermediate, is formed in a reaction that uses O.sub.2 and NADPH. Squalene epoxide is then cyclized to lanosterol by a cyclase. There is a concerted movement of electrons through four double bonds and a migration of two methyl groups in this remarkable closure. Finally, lanosterol is converted into cholesterol by the removal of three methyl groups, the reduction of one double bond by NADPH, and the migration of the other double bond.
From the foregoing, it is apparent that the mevalonate pathway is responsible for the synthesis of a wide variety of essential and clinically relevant molecules. Products from the mevalonate pathway include, for example, cholesterol and the prenyl groups which are required for the function of numerous small GTP binding proteins including the RAS oncoprotein. The connection of the mevalonate pathway, by virtue of its products, to many clinically important processes, such as atherosclerosis and RAS-based cancers, has led to a significant effort to understand the cellular regulation of the mevalonate pathway. It has been found that the mevalonate pathway is regulated by feedback control of several pathway enzymes, the principal one of which is 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol synthesis. Feedback regulation of HMG-CoA reductase occurs through modulation of the steady-state amount of protein by the coordinated adjustment of synthesis and degradation rates. Thus, when the mevalonate pathway products are abundant, the synthetic rate of HMG-CoA reductase is low, the degradation rate of HMG-COA reductase is rapid, and through both of these actions, the steady state of the enzyme is kept low. Conversely, when production of the mevalonate pathway products is slowed, the synthetic rate of the HMG-COA reductase is increased, the degradation rate of HMG-CoA reductase is slowed and, consequently, the steady-state level of HMG-COA reductase is elevated. To date, the molecular signals which control the synthesis and degradation of HMG-COA reductase are unknown. However, it is known that the control of HMG-CoA reductase degradation is genetically distinct from the control of HMG-CoA reductase synthesis, implying that distinct molecular mechanisms are responsible for these independent features of HMG-CoA reductase regulation.
The feedback regulation of the mevalonate pathway plays a central role in the clinical management of hypercholesterolemia. The current drugs of choice for lowering serum cholesterol are the structurally related, competitive inhibitors of HMG-CoA reductase which include, for example, the widely used Mevacor.RTM., the tradename for lovastatin. These agents were developed specifically for the purpose of inhibiting HMG-CoA reductase. HMG-CoA reductase was thought to be the best target for clinical control of the pathway because this enzyme is most strongly regulated in the physiological control of the cholesterol pathway. However, when a patient is put on an inhibitor of HMG-CoA reductase, the coordinated feedback regulation of HMG-CoA reductase synthesis and degradation brings about a compensatory increase of HMG-CoA reductase levels which abrogates most of the effect of the drug on body cholesterol synthesis. The beneficial effects of HMG-CoA reductase inhibitors, such as Mevaco.RTM., are thought to occur by increased clearance of plasma LDL brought about by the parallel up-regulation of the LDL receptor (LDL-R). Thus, the feedback signals from the mevalonate pathway severely limit the efficacy of such treatments on cholesterol synthesis, but, at the same time, are necessary for the beneficial effects of cholesterol clearing caused by drugs such as Mevacor.RTM..
From the foregoing, it is apparent that the potentially simple solution of interfering with the feedback signals from the mevalonate pathway in order to block the up-regulation of HMG-CoA reductase would also remove the beneficial increase of LDL receptors brought about by the same signals. Moreover, although the possibility exists that the detrimental up-regulation of HMG-CoA reductase synthesis could be specifically blocked without affecting the beneficial up-regulation of the LDL-R, the mechanistic similarity of the two processes may make this separation an unrealistic goal. As such, there remains a need in the art for appropriate molecular targets which can be used to manipulate the levels of HMG-CoA reductase in a manner which is independent of the beneficial LDL receptor control axis.
Another problem in the art is the function of many nucleic acid and polypeptide sequences on deposit in various sequence repositories. For example, one good system for studying the regulation of HMG-CoA is yeast, which encode an HMG-CoA reductase homologue. However, despite the fact that the entire yeast genome has been sequenced and the sequences deposited in GenBank.TM. and other sequence repositories, the relationship of many yeast genes to HMG-CoA reductase regulation is unknown. Elucidation of the complete yeast genome in the absence of functional information for a particular yeast gene is insufficient for identification of any particular gene product. Although many open reading frames (ORFS) have been identified, it is often not known whether these ORFs encode functional mRNAs, or what the function of the putative genes would be. Similarly, many eukaryotic sequences (e.g., human sequences) have been deposited in GenBank.TM. and other sequence repositories without any functional information for the sequences being available. In the absence of knowledge regarding the function of a given sequence, there is no reason to select the sequences for cloning into vectors, e.g., for expression of the nucleic acids and encoded proteins. In the absence of functional information, any relationship between deposited sequences and HMG-CoA reductase is unknown.